Phosphatidylinositide 3-kinases (PI 3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles. There are at least three genes in Dictyostelium discoideum, DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (Δddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM. However, Δddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme α-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed that Δddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1–3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells. Finally, Δddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization.

While autophagy is believed to be beneficial for life-span extension, it is controversial which forms or aspects of autophagy are responsible for this effect. We addressed this topic by analyzing the life span of yeast autophagy mutants under caloric restriction, a longevity manipulation. Surprisingly, we discovered that the majority of proteins involved in macroautophagy and several forms of microautophagy were dispensable for life-span extension. The only autophagy protein that is critical for life-span extension was Atg15, a lipase that is located in the endoplasmic reticulum (ER) and transported to vacuoles for disintegrating membranes of autophagic bodies. We further found that vacuole-vacuole fusion was required for life-span extension, which was indicated by the shortened life span of mutants missing proteins (ypt7Delta, nyv1Delta, vac8Delta) or lipids (erg6Delta) involved in fusion. Since a known function of vacuole-vacuole fusion is the maintenance of the vacuole membrane integrity, we analyzed aged vacuoles and discovered that aged cells had altered vacuolar morphology and accumulated autophagic bodies, suggesting that certain forms of autophagy do contribute to longevity. Like aged cells, erg6Delta accumulated autophagic bodies, which is likely caused by a defect in lipase instead of proteases due to the existence of multiple vacuolar proteases. Since macroautophagy is not blocked by erg6Delta, we propose that a new form of autophagy transports Atg15 via the fusion of vacuoles with vesicles derived from ER, and we designate this putative form of autophagy as secretophagy. Pending future biochemical studies, the concept of secretophagy may provide a mechanism for autophagy in life-span extension.

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